Compositions for selective reduction of circulating bioactive soluble tnf and methods for treating tnf-mediated disease

ABSTRACT

An isolated or synthetic antibody or ligand is provided that specifically binds to an epitope of a dissociated monomer of human TNF. Such binding disrupts assembly of the monomer into bioactive trimeric human sTNF. A pharmaceutical composition contains one or more antibodies or ligands: (a) an antibody or ligand that specifically binds the TNF monomer-specific epitope having the sequence PSDKPVAH or PSDKPVAHV, amino acids 8-15 or 8-16 of SEQ ID NO: 1; and (b) an antibody or ligand that specifically binds the TNF monomer-specific epitope having the sequence EPIYLGGVF, amino acids 116 to 124 of SEQ ID NO: 1. A combination of antibodies or ligands that bind or are reactive with (a) and/or (b) are useful in methods for treating a subject having a disease (e.g., rheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriatic arthritis, atherosclerosis, metabolic syndrome, Alzheimer&#39;s Disease, HIV, Type II diabetes) mediated by human TNF. These methods and compositions disrupt or reduce the in vivo assembly or reassembly of dissociated monomers of TNF into bioactive trimeric human sTNF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of pending U.S. patentapplication Ser. No. 15/176,503, filed on Jun. 8, 2016, which is adivision of U.S. patent application Ser. No. 14/695,551, filed on Apr.24, 2015, now U.S. Pat. No. 9,388,240, issued on Aug. 13, 2015, which isa continuation of international patent application No.PCT/US2014/012686, filed on Jan. 23, 2014, which claims priority of U.S.provisional patent application No. 61/768,044, filed Feb. 22, 2013 (nowexpired) and U.S. provisional patent application No. 61/756,571, filedJan. 25, 2013 (now expired). All patent applications are incorporatedherein by reference.

INCORPORATION-BY REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Applicant hereby incorporates by reference the Sequence Listing materialfiled in electronic form herewith. This file is labeledGGP11C1USA_ST25.txt”, was created on Apr. 21, 2015, and is 2 KB in size.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF; previously referred to as tumor necrosisfactor-α) is a proinflammatory cytokine that plays a major role in thepathogenesis of rheumatoid arthritis and associated inflammatorydiseases, such as ankylosing spondylitis, juvenile rheumatoid arthritis,and psoriatic arthritis. The human proform, transmembrane-bound TNF(tmTNF), is a 26-kDa homotrimer comprising three non-covalentlyassociated monomers, each monomer having N-terminal sequence imbedded inthe cell membrane. Each monomer of tmTNF has a 233 amino acid sequence(UniProtKB/Swiss-Prot entry Accession No. P01375). Soluble TNF (sTNF) isa homotrimer formed by enzymatic cleavage from its pro-form tmTNF. Eachmonomer of the sTNF trimer has a 157 amino acid sequence (SEQ ID NO: 1),which is the same sequence as aa77 to 233 of the published Acc No.P01374.

Both forms of active TNF (tmTNF and sTNF) exist as homotrimers¹⁰⁻¹³ andengage trimeric receptors that recognize receptor-binding sites in thegrooves between the TNF monomers in assembled homotrimers. The groovesbetween the monomers comprise amino acid sequence from two contiguousmonomers^(14,15). The receptor binding regions of both forms of TNF areidentical.

Trimer integrity is essential for biological function. For tmTNF,trimeric structure is established intracellularly before tmTNF insertioninto the cell membrane¹⁶ and is maintained in tmTNF by the anchoring ofthe protein stems passing through the membrane plus further lipidanchoring by palmitoylated amino acid side chains at the membraneboundary¹⁷. In contrast, sTNF active trimers dissociate freely intoinactive monomers SEQ ID NO: 1 and dimers that reform as active sTNFhomotrimers in a steady-state equilibrium between the three forms¹⁸.

Anti-TNF biologics have provided a major advance in the management ofthe above-noted inflammatory diseases with anti-TNF monoclonalantibodies REMICADE (Infliximab; Janssen Biotech, Inc.) and HUMIRA(Adalimumab, Abbott Laboratories), and a chimeric solubilized TNFreceptor fused to Fc, i.e., ENBREL (Entanercept, Biogen, Inc) beingwidely used^(1,2). This therapeutic and marketing success is marred bythe rare but statistically significant occurrence of serious infectionsand malignancies^(3,4), likely related to concomitant blockade oftmTNF^(5,6) function impairing immune defenses. These adverseoccurrences have included the development of tuberculosis, systemicfungal infection and other intracellular infections due principally tointracellular pathogens such as Mycobacterium tuberculosis, Listeriamonocytogenes and Histoplasma capsulatum, and certain forms of cancer.These results were unsurprising since these agents blockpro-inflammatory sTNF but also block tmTNF, which is essential forjuxtacrine cellular control of such intracellular infections andmalignancies^(3,7,9).

Because the receptor binding regions of both forms of TNF are identical,there has been little hope for the development of new monoclonalantibodies selectively blocking receptor engagements of one form versusthe other. Antibodies to short sequences of TNF have not lead to usefultherapeutics. For example, in 1987, Socher et al.²⁶ in exploringantibodies to full or partial synthetic sequences of TNF, observed ahigh polyclonal antibody response to the TNF fragment 1-15 that appearedto block bioactivity and receptor binding of TNF. However, this 16-yearold observation has not lead to the development of additionaltherapeutic reagents, likely because the TNF receptor is a discontinuoussurface region not associated with TNF amino acids 1-15. Subsequentresearchers in 2001 coupled TNF amino acids 4-23 conjugated topapillomavirus-like particles, and observed an induction of polyclonalantibodies, and an attenuation of experimental arthritis²⁷. Otherresearchers in 2007 used the same fragment TNF aa4-23 coupled to avirus-like particle-based composition and induced antibodies thatattenuated experimental arthritis. No suppression of resistance toinfection occurred, in contrast with full length TNF immunization²⁸.Because these TNF fragments were not directed to receptor bindingregions of TNF, these publications displayed no further teachings orsuggestion of therapeutic use of the resulting polyclonal antibodies;and further research has not been published since that date.

One more recent attempt to selectively suppress the pro-inflammatoryactivity of sTNF while preserving tmTNF function required for innateimmunity involved the design of synthetic dominant-negative TNF monomervariants that formed trimers that were inactive¹⁹. These were shown toattenuate experimental arthritis without suppressing innate immunity toinfection²⁰, emphasizing the major role of sTNF in pathogenesis ofarthritis. Another approach has been the search for small-molecule drugsthat interact with the inter-monomer contact regions. One molecule,SP304, bound such a contact region with μM affinity to effect trimerdisruption in vitro^(21,22).

Despite the plethora of literature in the field of anti-TNF treatmentfor a variety of inflammatory disorders, there remains a need in the artfor new and useful compositions and methods for generating therapeuticor prophylactic immunogenic compositions for these diseases which do notresult in adverse side effects due to suppression of cellular immunity.

SUMMARY OF THE INVENTION

As described herein the inventor has provided selective anti-TNFmonomer-specific biologic compositions and various methods of usethereof which do not affect the structure or bioactivity of tmTNF orincrease the treated subject's susceptibility to infection by anintracellular pathogen.

In one aspect, an isolated or synthetic antibody or ligand is providedthat specifically binds to an epitope of a dissociated monomer of humanTNF. The binding of the antibody or ligand to the monomer disrupts orprevents assembly of the monomer into bioactive trimeric human sTNF. Inone embodiment, the antibody or ligand binds specifically tomonomer-specific epitope A2 of sequence PSDKPVAH, amino acids 8-15 ofSEQ ID NO: 1 or PSDKPVAHV, amino acids 8-16 of SEQ ID NO: 1. In stillanother embodiment, the antibody or ligand binds specifically tomonomer-specific epitope F of sequence EPIYLGGVF, amino acids 116 to 124of SEQ ID NO: 1. In one embodiment, the antibody is a bi-specificantibody directed to epitopes A2 and F.

In another aspect, a pharmaceutical composition comprises one or moreisolated or synthetic antibody or ligand that specifically binds to anepitope of a dissociated monomer of human TNF, the binding disrupting orpreventing assembly of the monomer into bioactive trimeric human sTNF,and a pharmaceutically acceptable carrier or diluent. In certainembodiments, the composition contains one or two of the above-describedantibodies.

In yet a further aspect, methods for preparing or generating isolated orsynthetic antibodies or ligands that specifically bind to an epitope ofa dissociated monomer of human TNF are provided.

In still another aspect, a method for treating a subject having adisease mediated by soluble human TNF (sTNF) comprises reducing theamount, concentration or bioactivity of sTNF in the blood of a subjecthaving the disease without affecting the amount, concentration orbioactivity of tmTNF. This is accomplished by disrupting, preventing orreducing the in vivo assembly or reassembly of dissociated monomers ofTNF into bioactive trimeric human sTNF without affecting the amount,concentration or bioactivity of tmTNF. In certain embodiments, thismethod employs the monomer-specific antibodies, bi-specific antibodies,ligands and compositions described above and herein. In one embodiment,the disease is rheumatoid arthritis (RA), juvenile rheumatoid arthritis,ankylosing spondylitis (AS), psoriatic arthritis or psoriasis.

In still another aspect, sTNF elevations are also implicated in initialHIV infection, and the reoccurrence of latent HIV infection and type IIdiabetes. Therefore, in still another aspect, a method for preventing asubject infected with HIV-1 and treated with anti-retroviral drugs fromdeveloping a new infection (or rebound infection due to latent HIV)comprises administering to the subject treated with anti-retroviraltherapy (ART) with an isolated or synthetic selective anti-TNFmonomer-specific antibody or ligand, or pharmaceutical composition, asdescribed herein, after the ART is discontinued.

In yet another aspect, a method for treating a subject with type IIdiabetes comprises administering periodically to a subject in needthereof an isolated or synthetic selective anti-TNF monomer-specificantibody or ligand, or pharmaceutical composition, as described above,optionally in combination with known anti-diabetic therapies.

In other aspects, the TNF monomer-specific antibodies, ligands,bi-specific antibodies, or compositions described herein are providedfor use in the treatment of a disease or disorder mediated by solublehuman TNF, including any disease identified herein. In other aspects,use of the antibodies, ligands, or compositions described herein inpreparation of a medicament for treatment of a disease or disordermediated by soluble human TNF, including any disease identified herein,is provided.

Other aspects and advantages of these methods and compositions aredescribed further in the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the immunogen and epitope detector sequences for TNFinterface A and TNF Interface F, which were used to identify thesequences and margins of the three monomer-specific TNF epitopes A1, A2and F described herein as well as the binding activities of the anti-TNFmonomer-specific antibodies generated thereto. These sequences arediscussed in Example 1 below.

FIG. 2A is a bar graph illustrating the TNF monomer-specific epitopes byimmunizing a rat with sTNF amino acids 1-23. Antibody binding responseswere measured on recombinant TNF (rTNF), a mixture of monomers, dimersand trimers, and on synthetic peptides employing the indicatedterminally truncated TNF peptides. The synthetic detector peptidesincluded: TNF amino acids 1-10 of SEQ ID NO: 1, which demonstrated nobinding at all; TNF amino acids 5-12 of SEQ ID NO: 1, minimal to nobinding; the A1 epitope SSRTPSDKP, TNF amino acids 4-12 of SEQ ID NO: 1;TNF amino acids 4-11 of SEQ ID NO: 1 SSRTPSDK; TNF amino acids 9-15 ofSEQ ID NO: 1 SDKPVAH; the A2 epitope sequence of amino acids 8-15 of SEQID NO: 1; TNF amino acids 8-14 of SEQ ID NO: 1 PSDKPVA; and TNF aminoacids 16-23 of SEQ ID NO: 1 VVANPQAE, which exhibited no binding.Clearly only two overlapping epitopes were detected by rat antiserum toTNF amino acids 1-23, which were epitope A1, TNF amino acids 4-12 andA2, TNF amino acids 8-15.

FIG. 2B is a bar graph illustrating the margins of the A2 epitopePSDKPVAHV detected in mouse serum using the same procedure described inFIG. 2A, with synthetic peptides. The synthetic detector peptidesincluded: TNF amino acids 10-17 of SEQ ID NO: 1 DKPVAHVV, whichdemonstrated minimal binding; TNF amino acids 9-16, minimal binding; theA2 epitope PSDKPVAHV, TNF amino acids 8-16 of SEQ ID NO: 1; TNF aminoacids 8-15 of SEQ ID NO: 1 PSDKPVAH; and TNF amino acids 8-14 of SEQ IDNO: 1 PSDKPVA, no binding. When rat and rabbit sera were used, theboundaries of epitope A2 are amino acids 8-15 of SEQ ID NO: 1. Theimmune system of the mouse sees only TNF amino acids 8-16 and does notbind to TNF amino acids 8-15, as shown in the graph.

FIG. 2C is a bar graph illustrating the results of determining themargins of the monomer-specific TNF epitopes by immunizing a rat ormouse with sTNF amino acids 112-128 of SEQ ID NO: 1, KPWYEPIYLGGVFQLEK(interface region underlined; F epitope in bold). Antibody bindingresponses were measured on recombinant TNF (rTNF), a mixture ofmonomers, dimers and trimers, and on synthetic peptides employing theindicated four terminally truncated TNF peptides. The synthetic peptideswere IYLGGVF, amino acids 118-124 of SEQ ID NO: 1; PIYLGGVF, amino acids117-124 of SEQ ID NO: 1, EPIYLGGVF, amino acids 116-124 of SEQ ID NO: 1(epitope F) and EPIYLGGV, amino acids 116-123 of SEQ ID NO: 1. Thegreatest binding was to the aa116-124 peptide, thereby indicating themargins of the epitope (referred to as epitope F).

FIG. 3A illustrates data from the sandwich assay using 200 ng/mL sTNF,biotin-labeled antibody and non-biotin labeled antibody, as described inExample 2 below. The binding curves show that commercial REMICADEanti-TNF antibody, when labeled with biotin and mixed with sTNF(trimers, dimers and monomers), binds multimeric forms of TNF. Thebiotinylated antibody-TNF in the mixture still has available TNF trimerepitopes that can bind and form a sandwich with the unlabeled platedREMICADE antibody (). In contrast, Protein A/Protein G purified IgG (◯)was obtained from rats immunized with TNF amino acids 1-23 of SEQ IDNO: 1. This purified IgG contains a mixture of monomer-specific anti-TNFthat selectively bind epitope A1 and monomer-specific anti-TNF thatselectively bind epitope A2. The purified IgG (◯) does not sandwich inthe assay, because once these antibodies bind the TNF monomers in theTNF mixture, the labeled monomer-specific antibody-TNF complexes have noavailable monomer-specific epitopes to bind to the plated unlabeledmonomer-specific antibody on the plate. Labeled TNF monomer-specificantibody-monomers complexes are simply washed from the plate withoutbinding. REMICADE antibody that binds trimeric TNF was used as apositive control in this assay. Thus neither anti-A1 nor anti-A2antibodies bind the trimeric form of sTNF.

FIG. 3B illustrates data from a similar sandwich assay to that of FIG.3A, using as reagents: commercial REMICADE anti-TNF antibody ();affinity purified IgG from monomer-specific antisera to the TNF epitopeF that selectively binds only epitope F: EPIYLGGVF (X) and Protein A/Gpurified IgG from monomer-specific antisera to the TNF epitope F thatselectively binds only epitope F: EPIYLGGVF (□). Thus, in contrast tothe commercial REMICADE anti-TNF antibodies, the anti-F antibodies donot bind the trimeric form of sTNF.

FIG. 4A illustrates the results of an assay of antibody inhibition ofsTNF-induced cytotoxicity in target cells, using antiserum generated toTNF amino acids 1-23 that contains antibodies that selectively bind themonomer specific epitopes PSDKPVAH and SSRTPSDKP (epitopes A2 and A1,respectively). The titers of antisera with 200 pg/mL TNF in all wellsare displayed under the bars, from 1×10⁶, 2.5×10⁵, 1×10⁵, 2.5×10⁴,1×10⁴, and 2.5×10³. The last bar is sTNF in 50% NRS.

FIG. 4B illustrates functional blocking of sTNF cytotoxicity inactinomycin-treated WEHI cells in the assay described in Example 2 byantiserum to TNF epitope F. The indicated dilutions (50%, 16.7%, 5.6%,1.85%, 0.48%) of monomer-specific antisera generated to the immunogenKPWYEPIYLGGVFQLEK, amino acids 112-128 of SEQ ID NO: 1 (the F beta sheetinterface sequence of TNF), in rats were compared for their ability toinhibit sTNF bioactivity with 200 pg/mL TNF and 50% NRS (normal ratserum). As is shown in this figure, inhibition of TNF cytotoxicity wasshown using antiserum diluted from 0.48% to 50%. Thus, themonomer-specific antisera to epitope F showed the ability to inhibit thecytotoxic effect of sTNF on the cells as evidenced by increasingreplication of cells in the presence of the antisera. Statisticalsignificance was determined by one-way ANOVA and post testing withDunnett's test. REMICADE antibody, which binds trimeric TNF, was used asa positive control in this assay.

FIG. 5 is a bar graph showing inhibition of sTNF cytotoxicity in WEHIcells by monoclonal antibody generated to TNF epitopes A1 or A2. Cellreplication (OD) was measured in WEHI cells grown in the presence of noTNF and the cells showed good replication. WEHI cells grown in thepresence of 0.2 ng/ml full length TNF 1-157 of SEQ ID NO: 1 demonstratedthat TNF inhibited replication. Cells grown in TNF plus the commercialREMICADE anti-TNF antibody at 1 μg/mL, showed that the antibody returnedreplication to the same levels demonstrated in the absence of TNF. TNFplus the inventor's monoclonal monomer-specific antibody A1-4H6 to TNFepitope A1 (amino acids 4-12 of SEQ ID NO: 1) at 10 μg/mL, failed toinhibit WEHI cell replication, which remained at the levels of cellsexposed to TNF alone. WEHI cells were cultured in the presence of TNFplus the inventor's monomer-specific monoclonal antibody A2-8D12 to TNFepitope A2 (amino acids 8-16 of SEQ ID NO: 1) at 10 μg/mL; and TNF plusthe inventor's monomer-specific monoclonal antibody A2-10H10 to TNFepitope A2 (amino acids 8-16 of SEQ ID NO: 1) at 0.25 μg/mL. Data fromthese two latter monomer-specific monoclonal antibodies showed highlystatistically significant inhibition of sTNF cytotoxicity (i.e.,reduction of cell killing). The second monomer-specific monoclonalantibody A2-10H10 showed high sensitivity, a 40× increase in potency inthis assay over the other monomer-specific anti-A2 antibody A2-8D12.These results demonstrate that monomer-specific anti-A2 monoclonalantibodies inhibit sTNF and that one such antibody A2-10H10 exhibits ahigher affinity than the other, as demonstrated by the 40 fold lowerdose. No inhibition even at a high dose was demonstrated by themonomer-specific anti-A1 monoclonal antibody. These data demonstratethat the effects of the TNF amino acid 1-23 polyclonal antisera were dueto the monomer-specific anti-A2 antibodies only.

DETAILED DESCRIPTION OF THE INVENTION

The inventor has provided selective anti-TNF monomer-specific biologiccompositions and various methods of use based on the determination thatantibodies and/or other ligands directed to selected epitopes partiallyor fully within the internal interface contact region of TNF freemonomers block their association with other monomers and causeprogressive disruption of bioactive sTNF trimer formation. It isadvantageous to have an antibody or ligand, e.g., a monoclonal antibodyor bi-specific antibody, that selectively blocks the activity of sTNFbut not tmTNF for the treatment of rheumatoid arthritis (RA), juvenilerheumatoid arthritis, ankylosing spondylitis (AS) and psoriaticarthritis (PA), psoriasis, and other inflammatory diseases.

I. ANTIBODIES/LIGANDS

Thus, this invention provides an isolated or synthetic antibody orligand that specifically binds to an epitope of a dissociated monomer ofhuman TNF, the binding disrupting or preventing assembly of monomersinto bioactive trimeric human sTNF.

The inventor determined that antibodies directed to certain epitopespartially or fully within the internal interface contact region of freeTNF monomers would block their association with other monomers and causeprogressive disruption of trimer formation. In contrast to the knownpublications on TNF, the inventor determined that there were twooverlapping epitopes in the TNF sequence of amino acids 1-15 of SEQ IDNO: 1. One epitope A1 which spanned amino acids 4-12 of SEQ ID NO: 1 wasmonomer-specific, but did not disrupt trimer formation. The otherepitope A2, which spanned amino acids 8-15 or 8-16 of SEQ ID NO: 1, wasmonomer-specific and did disrupt trimer formation. Further the inventoridentified a new unrecognized epitope F, amino acids 116-124 of SEQ IDNO: 1 in the F β sheet of TNF, which was monomer-specific and diddisrupt trimer formation. The discovery and new uses of antibodies orligands that specifically bind these epitopes is discussed in detailbelow and in the examples.

Early x-ray crystallography studies established that the 157 amino acid(SEQ ID NO: 1) sTNF monomers formed an “elongated, anti-parallel βpleated sheet sandwich with “jelly-roll” topology”. Three monomers inintimate but non-covalent association constituted the active trimer₁₁.Five stretches of amino acid sequences formed the interface β sheetcontact surfaces: A, aa11-18; A′, aa35-39; C, aa54-67; F, aa114-126 andH, aa149-157, all of SEQ ID NO: 1, where A, A′, C, F and H refer to a (3sheet naming convention¹¹. The inventor explored all five regions forpotential B cell epitopes and detected and mapped antibodies to twoepitopes partially (the A β sheet) and one epitope fully (the F β sheet)within an interface region, all being outside the known regions of thereceptor binding sites of TNF^(12,13,15) See, Example 1 below.

In one embodiment, the selective anti-TNF monomer-specific antibody orligand binds the epitope A2 of sequence PSDKPVAH, amino acids 8-15 ofSEQ ID NO: 1 or sequence PSDKPVAHV, amino acids 8-16 of SEQ ID NO: 1. Inanother embodiment a selective anti-TNF monomer-specific antibody orligand binds the A1 epitope of sequence SSRTPSDKP, amino acids 4-12 ofSEQ ID NO: 1. In another embodiment, the TNF monomer-specific antibodyor ligand binds the F epitope of sequence EPIYLGGVF, amino acids 116 to124 of SEQ ID NO: 1.

As described below, antibodies to A1 epitope, while TNFmonomer-specific, have been found to be inactive in blocking TNFfunction when used alone. Antibodies to these A2 and F epitopesspecifically bind TNF monomers but not trimers, disrupt assembly of sTNFtrimers and inhibit sTNF function in vitro. These monomer-specificanti-A2 and anti-F epitope antibodies or ligands do not bindtransmembrane TNF (tmTNF) and do not affect the structure or bioactivityof tmTNF. Additionally, these antibodies or ligands do not bind intactbioactive trimeric human sTNF. Monoclonal antibodies to the A2 and Fepitopes are useful for therapeutic use as safer sTNF-selective anti-TNFbiologics for rheumatoid arthritis and related inflammatory diseaseswith sTNF-related pathologies.

As used herein, the term “antibody” refers to an intact immunoglobulinhaving two light and two heavy chains. The term “antibody fragment”refers to less than an intact antibody structure, including, withoutlimitation, an isolated single antibody chain, an sc-Fv construct, a Fabconstruct, a Fab₂ construct, or a light chain variable orcomplementarity determining region (CDR) sequence, etc. The term“bi-specific” antibody refers to a synthetically or recombinantlyproduced antibody that contains one heavy and/or one light chain thatbinds to one epitope, e.g., the A2 epitope, and one heavy and/or onelight chain that binds to a second epitope, e.g., the F epitope. Theterm “ligand” is used to refer to other synthetic molecules or sequencesthat can be designed to bind to the indicated epitopes.

“High affinity” is the strength of binding of the antibody or ligand inquestion to the TNF monomer-specific epitope A2 or F. In one embodiment,the antibodies/ligands to A2 or F bind at an affinity of less than 10nanomolar (nM). In another embodiment, the antibodies/ligands to A2 or Fbind at an affinity of less than 1 nanomolar (nM). In anotherembodiment, the antibodies/ligands to A2 or F bind at an affinity ofless than 100 picomolar (pM). In another embodiment, theantibodies/ligands to A2 or F bind at an affinity of less than 10 pM.

Thus, in one embodiment, the antibody or ligand as described herein maybe a polyclonal, affinity-purified or high affinity antibody or afragment thereof. In one embodiment, the antibody or ligand is amonoclonal antibody or a fragment thereof. In another embodiment, theantibody or ligand is an isolated single chain of an antibody. Stillother forms of antibodies, such as a synthetic antibody, a recombinantantibody, a chimeric antibody, a humanized antibody, a human antibody ora fragment thereof can be employed as the ligand or antibody directed toone of the above described epitopes. Suitable fragments of suchantibodies may also be employed. In yet another embodiment, the antibodyor ligand or fragment thereof further comprises a polyethylene glycol(PEG) molecule. The antibody or ligand or fragment can be associated orfused with PEG by known conventional methodologies.^(30,31)

The production of antibodies or ligands that specifically bind to one ofthe selected epitopes, can employ conventional techniques. For example,polyclonal antibody compositions are typically produced by immunizing aselected mammal, e.g., a primate, rodent, or human, with apeptide/polypeptide composition containing a specific epitope. See,e.g., the description of the antisera described in FIGS. 2A-2C. Theselection of high titer, high affinity polyclonal antibodies can bemonitored by standard techniques, such as with an enzyme-linkedimmunosorbent assay and surface plasma resonance. If desired, thepolyclonal antibody molecules can be isolated from the mammal, e.g.,from the whole blood, plasma or serum, and further purified from theplasma or serum of the immunized mammal by conventional techniques.Conventional harvesting techniques can include plasmapheresis, proteinA/G chromatography, among others. Such polyclonal antibody compositionsmay themselves be employed as pharmaceutical compositions of thisinvention.

Alternatively, monoclonal antibodies can be generated to any one of theepitopes by now conventional techniques, using antibody producing cellsobtained from the immunized mammals and fused to non-IgG-producingmyeloma cells to form hybridomas or from selection from activated immuneB cells with extraction by known molecular biological techniques. Thesemonoclonal antibodies can be further used to prepare other forms ofantibodies and ligands, e.g., chimeric antibodies, humanized antibodies,human antibodies. Other antibody fragments or ligands can be produced byscreening phage display libraries, antibody fragments and mixturesthereof. Techniques for generating these types of antibodies and ligandsare well-known in the art and the ligands themselves may be generatedusing the disclosed amino acid sequences of the above-identifiedepitopes.^(32,35-39)

Chimeric antibodies may similarly be developed using known techniques.Chimeric antibodies are molecules in which different portions arederived from different animal species. Single chain antibodies may alsobe prepared by conventional methods, such as described in U.S. Pat. Nos.4,946,778 and 4,704,692 using the variable portions of the polyclonal ormonoclonal antibodies produced according to this invention. Antibodyfragments, such as the Fab, F(ab)₂ and scFv fragments and librariesthereof may also be employed in generation of the selective anti-TNFmonomer-specific antibodies or ligands as described herein.

The production of bi-specific antibodies or ligands that specificallybind to two or more of the selected epitopes, can employ conventionaltechniques. It is within the skill of the art to develop bi-specificantibodies that bind multiple epitopes. See, e.g., Hornig N,Färber-Schwarz A., “Production of bispecific antibodies: diabodies andtandem scFv.”, 2012, Methods Mol Biol., 907:713-27; Speiss, C. et al,“Bispecific antibodies with natural architecture produced by co-cultureof bacteria expressing two distinct half-antibodies, Jul. 7, 2013,Nature Biotechnology, 31:753-758; and Jonathan S Martin and ZhenpingZhu, “Recombinant approaches to IgG-like bispecific antibodies”, 2005Acta Pharmacologica Sinica, 26: 649-658. In one embodiment, a bispecificantibody is developed which is capable of binding to or reacting withepitope A2 and epitope F. It is anticipated that such bispecificantibodies, e.g., the antibody reactive with A2 and F, will enhanceavidity and create greater potency than the single anti-A2 or anti-Fantibodies or ligands alone.

Other selective anti-TNF monomer-specific antibodies or ligands may bedeveloped by screening recombinant combinatorial immunoglobulin scFvlibraries (e.g., phage displays) with one of the above-identified TNFmonomer-specific epitopes to isolate immunoglobulin library members thatbind to the TNF monomer. See, e.g., Phage Display of Peptides andProteins, A Laboratory Manual, eds. Kay, B K et al, Elsevier Inc.(1996), among other texts well known in the art. Kits for generating andscreening phage display libraries are commercially available, e.g.,Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01;Stratagene Phage Display kits, etc. See, e.g., U.S. Pat. No. 5,223,409,International Publication No. WO92/09690, WO90/02809, etc.

II. PHARMACEUTICAL COMPOSITIONS

In another aspect, a pharmaceutical composition comprises an isolated orsynthetic antibody or ligand that specifically binds to an epitope of adissociated monomer of human TNF, the binding disrupting or preventingassembly of the monomer into bioactive trimeric human sTNF.

The pharmaceutical composition contains one or more of the selectiveanti-TNF monomer-specific antibodies or ligands described above with asuitable carrier or diluent. Thus, in one embodiment, the pharmaceuticalcomposition contains an anti-TNF monomer-specific antibody or ligandthat specifically binds the A2 epitope having the sequence PSDKPVAH,amino acids 8-15 of SEQ ID NO: 1 or PSDKPVAHV, amino acids 8-16 of SEQID NO: 1. In still another embodiment, the pharmaceutical compositioncontains a selective anti-TNF monomer-specific antibody or ligand thatspecifically binds the F epitope having the sequence EPIYLGGVF, aminoacids 116 to 124 of SEQ ID NO: 1.

In another embodiment, a pharmaceutical composition comprises twoanti-TNF monomer-specific antibodies or ligands. In one embodiment, thecomposition comprises an antibody or ligand that specifically binds theA2 epitope PSDKPVAH, amino acids 8-15 of SEQ ID NO: 1 or PSDKVPAHV,amino acids 8-16 of SEQ ID NO: 1 and an antibody or ligand thatspecifically binds the F epitope having the sequence EPIYLGGVF.

In yet a further embodiment, a pharmaceutical composition containing abispecific antibody that specifically binds with the A2 epitope and theF epitope is also useful in interfering with soluble TNF trimerformation. Other forms of multi-ligand constructs known to the art mayalso take advantage of binding to A2 and/or F epitopes to provide trimerdisruption. Alternatively, the anti-TNF monomer-specific compositions ofthis invention may be used in conjunction with, or sequentially with,other therapies or pharmaceutical regimens which are used conventionallyto treat the various diseases mediated by sTNF.

These pharmaceutical compositions described herein also contain one ormore pharmaceutically acceptable carriers or diluents. As definedherein, the pharmaceutically acceptable carrier suitable for use in animmunogenic proteinaceous composition of the invention are well known tothose of skill in the art. Such carriers include, without limitation,water, saline, buffered saline, phosphate buffer, alcoholic/aqueoussolutions, emulsions or suspensions. Other conventionally employeddiluents, adjuvants and excipients, may be added in accordance withconventional techniques. Such carriers can include ethanol, polyols, andsuitable mixtures thereof, vegetable oils, and injectable organicesters. Buffers and pH adjusting agents may also be employed. Buffersinclude, without limitation, salts prepared from an organic acid orbase. Representative buffers include, without limitation, organic acidsalts, such as salts of citric acid, e.g., citrates, ascorbic acid,gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid, Tris, trimethanamine hydrochloride, or phosphatebuffers. Parenteral carriers can include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's orfixed oils. Intravenous carriers can include fluid and nutrientreplenishers, electrolyte replenishers, such as those based on Ringer'sdextrose and the like. Preservatives and other additives such as, forexample, antimicrobials, antioxidants, chelating agents, inert gases andthe like may also be provided in the pharmaceutical carriers. Thepresent invention is not limited by the selection of the carrier. Thepreparation of these pharmaceutically acceptable compositions, from theabove-described components, having appropriate pH isotonicity, stabilityand other conventional characteristics is within the skill of the art.See, e.g., texts such as Remington: The Science and Practice ofPharmacy, 22nd ed, Lippincott Williams & Wilkins, publ., 2012; and TheHandbook of Pharmaceutical Excipients, 7th edit., eds. R. C. Rowe et al,Pharmaceutical Press, 2012.

III. METHODS OF USE

A. Treatment of Certain Inflammatory Conditions

A method for treating a mammalian, preferably human, subject having adisease mediated by soluble human TNF (sTNF) involves reducing theamount, concentration or bioactivity of sTNF in the blood of a subjecthaving the disease without affecting the amount, concentration orbioactivity of tmTNF. This reduction occurs by disrupting, preventing orreducing the in vivo assembly or reassembly of dissociated monomers ofTNF into bioactive trimeric human sTNF. Thus, in one embodiment, thismethod comprises administering to a subject in need thereof an isolatedor synthetic anti-TNF monomer-specific antibody or ligand thatspecifically binds to an epitope of a dissociated monomer of TNF. Theselected antibodies or ligands do not bind intact bioactive sTNF trimer.The selected antibody or ligand does not bind transmembrane TNF (tmTNF)and does not affect the structure or bioactivity of tmTNF.

In one embodiment, the method is useful for the treatment of rheumatoidarthritis. In another embodiment, the method is useful for the treatmentof ankylosing spondylitis. In another embodiment, the method is usefulfor the treatment of juvenile rheumatoid arthritis. In still anotherembodiment, the method is useful for the treatment of psoriaticarthritis. In still another embodiment, the method is useful for thetreatment of psoriasis. In another embodiment, the method is useful forthe treatment of a pathogenic effect of bioactive, trimeric sTNFproduced during inflammation or during the course of an inflammatorydisorder. Still additional embodiments of the methods of the inventioninvolve treatment of other diseases in which sTNF and/or inflammation atlow or chronic levels plays a role. In one embodiment, such a disease isHIV-1. In another embodiment, the methods are useful for treating type 2diabetes. In still other embodiment, the therapeutic selective anti-TNFmonomer-specific antibodies or ligands are useful in methods fortreating inflammation in the pathology of obesity. In anotherembodiment, the method is useful for the treatment of metabolicsyndrome. In another embodiment, the method is useful for the treatmentof atherosclerosis and associated cardiovascular disease. In anotherembodiment, the method is useful for the treatment of inflammationinvolved in the pathology of Alzheimer's disease. In another embodiment,the method is useful for the treatment of inflammation involved in thepathology of neurodegenerative diseases. Still other inflammatorydiseases⁴⁰ may be treated with the compositions and methods describedherein. Such treatment is not burdened by the immune suppression andmorbidity and mortality associated with non-selective agents.

Therefore, in one embodiment the antibody/ligand useful in the methodbinds the A2 epitope sequence PSDKPVAH or PSDKPVAHV. In anotherembodiment of the method, the antibody/ligand useful in the method bindsthe F epitope EPIYLGGVF. The binding of the antibody/ligand to theseselected epitopes disrupts or prevents assembly of the monomer intobioactive trimeric human sTNF. In still further embodiments of thismethod, the subject is administered two of these selective anti-TNFmonomer-specific antibodies/ligands. In still other embodiments, thepharmaceutical compositions may include the bispecific antibodiesdiscussed above.

Another aspect of this method involves maintaining a reduced amount orconcentration of bioactive trimeric sTNF in the subject's bloodstreamover time. Such maintenance can involve repeated administration of oneof more of the above-noted selective anti-TNF monomer-specificantibodies, ligands, monoclonal antibodies, bispecific antibodies orpharmaceutical compositions containing same. By use of these methods,the subject's susceptibility to infection by an intracellular pathogen,e.g., tuberculosis, bacterial sepsis, invasive fungal infection, orhistoplasmosis, or to a malignancy, e.g., lymphoma or hepatosplenicT-cell lymphoma, is not increased by treatment.

According to these therapeutic methods, the selective anti-TNFmonomer-specific antibody or ligand is present in a pharmaceuticalcomposition in a pharmaceutically acceptable carrier or diluent. Any ofthe pharmaceutical compositions described above, e.g., containing one ortwo of the antibodies/ligands, and possibly antibodies directed tonon-sTNF immunogens, can be employed.

In each of the above-described methods, these compositions of thepresent invention are administered by an appropriate route, e.g., by thesubcutaneous, mucosal, intravenous, intraperitoneal, intramuscular,nasal, or inhalation routes. The presently preferred route ofadministration is subcutaneous, intravenous or intramuscular.

The amount of the selective anti-TNF monomer-specific antibody, ligand,monoclonal or bispecific antibodies, or constructs described above, withor without other antibodies or ligands to other immunogens, present ineach dose, is selected with regard to consideration of the patient'sage, weight, sex, general physical condition and the specific diseasebeing treated. The amount of antibody required to produce an exogenouseffect in the patient without significant adverse side effects variesdepending upon the pharmaceutical composition employed. In patients witha disease medicated by sTNF, generally, each dose will comprise betweenabout 5 to 400 mg/mL injection of the selective anti-TNFmonomer-specific antibody in a sterile solution. Another dosage is about200 mg/mL of the antibody. Still another dosage is about 100 mg/mL ofthe antibody. Still another embodiment is a dosage of about 50 mg/mL ofthe antibody. A further embodiment is a dosage of about 10 mg/mL of theantibody. When used together, dosages of each anti-TNF monomer-specificantibody to a different one of the two monomer-specific TNF epitopes maybe the same. In another embodiment, due to the synergy between the twocombined selected anti-TNF monomer-specific antibodies, a combinationdosage is lower than additive single dosages of each antibody alone. Forexample, the dosage of a bi-specific antibody directed to A2 and F maybe less than the dosage of an antibody to one of these epitopes alone.Additional combination with antibodies directed to other than sTNFepitopes may alter the dosage of the anti-TNF monomer-specificantibodies.

In one embodiment, the administration of the selective anti-TNFmonomer-specific antibody/ligand is repeated periodically during thecourse of the disease. In various embodiments in which two of theselective anti-TNF monomer-specific antibodies are administered in thecourse of treatment, each antibody/ligand in a pharmaceuticallyacceptable carrier is administered, either separately, in combination,or sequentially in any order.

The frequency of administration may range from weekly to monthly orbimonthly, and less frequently, and may depend upon the half-life of theantibody and the course of the particular disease. In one embodiment,the dosage is administered once a week. In another embodiment, thedosage is administered once every two weeks. In another embodiment thedosage is administered once a month. In another embodiment, thefrequency of dosage administration is once every two or three months.Other dosage ranges may also be contemplated by one of skill in the art,particularly where administration of the antibody composition is inconjunction or sequential with other treatments for the disease.

B. Treatment of HIV-1 Infection

In still another aspect, a method for treating a subject to reduce orprevent re-infection or rebound infection with latent HIV-1 in a subjecttreated with anti-retroviral drugs is provided. According to thismethod, a subject receiving ART is administered the isolated orsynthetic selective anti-TNF monomer-specific antibody or ligand,monoclonal antibody or bispecific antibody to epitopes A2 or F, orpharmaceutical composition containing same. In one embodiment, ananti-TNF monomer-specific antibody that binds A2 or F epitope isadministered to a subject receiving anti-retroviral therapy. In anotherembodiment, an anti-TNF monomer-specific antibody or ligand orcomposition that binds A2 or F epitope is administered to a subjectstarting immediately after ART is discontinued. In still anotherembodiment, the anti-TNF monomer-specificantibodies/ligands/compositions are administered chronically to asubject both before ART treatment is discontinued and chronically afterART treatment is discontinued. The purpose of this method of treatmentis to prevent re-infection or rebound infection of the subject with adifferent latent strain or variant of HIV after ART has successfullycontrolled the initial HIV infection.

In still other embodiments, the selective anti-TNF monomer-specificantibodies/ligands may be used in concert with other anti-HIVcompositions (see e.g., U.S. Pat. No. 7,943,140).

According to any of the above methods, the selective anti-TNFmonomer-specific antibody/ligands are administered in a pharmaceuticallyacceptable carrier, either separately, in combination, or sequentiallyin any order. The compositions, dosages, routes of administration andfrequency of administration are anticipated to be as described. However,one of skill in the art, given the teachings of this application mayemploy other suitable dosages and routes of administration. Particularlyfor HIV, the administration of the selective anti-TNF monomer-specificantibody/ligand/compositions is expected to be repeated periodically foran indefinite period.

C. Treatment of Diabetes

In still another aspect, sTNF elevations are also implicated in type IIdiabetes. Yet a further embodiment of this invention involves a methodfor treating diabetes which may also be practiced utilizing theselective anti-TNF monomer-specific antibodies and pharmaceuticalcompositions described herein. In one embodiment, this method fortreating a human subject with type II diabetes comprises administeringperiodically to a subject in need thereof an isolated or syntheticselective anti-TNF monomer-specific antibody or ligand, orpharmaceutical composition. The antibody, ligand or pharmaceuticalcomposition may be any of those described specifically above. In oneembodiment, the subject is concurrently treated with other diabetesmedication. In still another embodiment, the administration of theselective anti-TNF monomer-specific antibody/ligand or composition isrepeated periodically after the subject ceases treatment with otherdiabetes medications, such as insulin or oral drugs such as metformin.

IV. EXAMPLES

The following examples illustrate certain embodiments of theabove-discussed compositions and methods. These examples do not limitthe disclosure of the claims and specification.

Example 1—TNF Epitope Mapping

The 157 amino acid TNF monomers SEQ ID NO: 1 have an elongated,anti-parallel β pleated sheet structure. When three monomers areassociated in a non-covalent trimer, bioactive sTNF is formed. Fivestretches of amino acid sequences form the interface β sheet contactsurfaces:

A—KPVAHVVA, aa11-18 of SEQ ID NO: 1;

A′—ALLAN, aa35-39 of SEQ ID NO: 1;

C—GLYLIYSQVLFKGQ, aa54-67 of SEQ ID NO: 1;

F—WYEPIYLGGVFQL, aa114-126 of SEQ ID NO: 1; and

H—QVYFGIIAL, aa149-157 of SEQ ID NO: 1,

where A, A′, C, F and H refer to a β sheet naming convention¹¹.

To attain trimer disruption immunologically, the inventor theorized thatantibody binding to epitope sequences that are wholly or partiallywithin the contact area between adjacent monomers (the so-calledinternal or interface regions) would not bind to intact trimers of sTNFor tmTNF but would only bind to free monomers of TNF. In binding only tothe free monomers, these antibodies would disrupt or prevent the abilityof the monomers to re-associate and form active trimers.

Therefore, the inventor explored all five regions for potential B cellepitopes and detected and mapped antibodies to two epitopes partially(the A interface β sheet contact surface) and one epitope fully (the Finterface β sheet contact surface) within an interface region. Rats ormice were immunized with synthetic peptides derived from the linearsequence from the five known interface regions of TNF identified above⁵.These synthetic peptides sequences were conjugated with KLH, andadjuvants such as Freunds Complete or Incomplete Adjuvant were used.Alternatively the synthetic peptide sequences are incorporated in selfadjuvanting constructs, such as those described for HIV Tatconstructs.³³ Polyclonal antibodies isolated from rats immunized witheach of the synthetic peptide sequences were evaluated on rTNF and alsoon the synthetic TNF peptides using conventional ELISAs. See, e.g., theprotocols described for anti-human TNF/TNFSF1A antibody by R&D Systems,catalog number MAB610, clones 28401, pages 1 and 2 (Jun. 17, 2005).

Binding to truncated sequences from larger peptide immunogens was usedto delineate epitope margins. When antibodies were detected to a regionof TNF, truncated peptide sequences were used to determine the marginsof the epitopes defined above. An antibody that bound exclusively to oneof each the specific epitope sequences was referred to as a selectiveanti-TNF monomer-specific antibody of this invention. See, e.g., FIG. 1.

Table 1 sets out the interface regions, immunogens tested and epitopesdetected from the epitope searches:

TABLE 1 Immunogens Interface of SEQ ID Region NO: 1 TestedEpitopes Detected* Titers A aa1-15 and aa4-12: SSRTPSDKP (epitope A1)400,000 to 1 million aa1-24 aa8-15: PSDKPVAH (epitope A2) A′ aa35-39No significant antibody response — C aa52-67No significant antibody response — F aa112-128aa116-124:EPIYLGGVF (epitope F)  50,000 to 100,000 H aa145-157No significant antibody response — Bold amino acids within interfacesequences

FIGS. 2A, 2B and 2C illustrate the margin identification of theepitopes.

All three epitope sequences are outside of the known regions of thereceptor binding sites of TNF^(12,13,15). The two overlapping B cellepitopes detected in synthetic peptide sequences overlapping interfacecontact region A are:

PSDKPVAH, amino acids 8-15 of SEQ ID NO: 1 and PSDKPVAHV, amino acids8-16 of SEQ ID NO: 1; and

SSRTPSDKP, amino acids 4-12 of SEQ ID NO: 1.

Thus, the sTNF aa1-23 sequence of SEQ ID NO: 1 contains only two B cellepitopes, each epitope containing interface amino acid(s) essential forantibody binding, but only epitope A2 contains amino acids necessary fortrimer formation and biological function.

The entire sequence of the third epitope EPIYLGGVF (F sheet), aminoacids 116-124 of SEQ ID NO: 1 was within interface contact region F. Twoof the epitope amino acids, Tyr₁₁₉ and Gly₁₂₂ are critical for trimerformation and biological activity^(13,24).

In the three epitopes shown above, the bolded amino acids are within theinternal interface regions. The italicized amino acids are those atwhich point mutations induce failure to form trimers and loss ofbiological activity.

Antibodies to each of three monomer-specific epitopes A1: SSRTPSDKP, A2:PSDKPVAH/PSDKVPAHV and F: EPIYLGGVF bind to synthetic rTNF (whichcomprises trimer, dimer and monomer forms) in the conventional ELISAassay. Antibodies to A1 SSRTPSDKP (both polyclonal antibodies andmonoclonal antibodies) bound only two interface amino acids (Lys₁₁ andPro₁₂), essential for antibody binding to the epitope. Neither of theseamino acids have been shown to be critical for trimer formation andbioactivity of sTNF. Antibodies to A2 PSDKPVAH or PSDKPVAHV (bothpolyclonal antibodies and monoclonal antibodies) also masked His₁₅ thatis critical to trimer formation and bioactivity of sTNFmolecules^(13,23).

Only one other monomer specific antibody was detected, within the Fmonomer interface region. The F epitope spanned TNF amino acids 116-124and induced polyclonal antibodies that were monomer-specific. Theseantibodies masked Tyr₁₁₉ and Gly₁₂₂, both critical to trimer formationand bioactivity of sTNF molecules.

Example 2—Selective Binding Activity

Natural or synthetic sTNF consists of a mixture of inactive TNFmonomers, inactive TNF dimers and bioactive TNF trimers. Each antibodyused in the assays and commercially available anti-TNF antibodies (e.g.,REMICADE) or commercially available TNF receptor chimera (e.g., ENBREL)binds to synthetic TNF coated on a plate, as in the conventional ELISAsperformed in Example 1. However, detection of specific binding of anantibody/ligand to the monomeric, dimeric or trimeric form of sTNFrequires appropriate selective assays. To demonstrate the selectivebinding activity of antibodies or ligands that bind only the threeepitopes on a monomer as identified in Example 1, the following assaysare performed:

A. Sandwich Assay

In one embodiment, a sandwich assay employs a biotinylated anti-TNFantibody which binds the sTNF in a sample, followed by detection withthe same antibody, non-biotinylated, coated on a plate²⁵. The unlabeledantibody is plated (e.g., one of the inventor's epitope-bindingantibodies or a commercial antibody, e.g., REMICADE antibody). A samplecontaining synthetic sTNF mixed with the same anti-TNF antibody, whichhas been biotinylated, is prepared and then introduced to the plate. Theplate is then washed and any bound sTNF sandwiched between the unlabeledbound antibody and the labeled detector antibody is measured using asuitable detector system, e.g., streptavidin/horseradish peroxidase, togenerate a detectable signal.

If the antibody used as the capture/detector antibody is an antibodythat binds sTNF trimeric form, a sandwich effect will be detected. OnlysTNF trimers with the ability to simultaneously bind both theplate-bound antibody and the biotinylated antibody will demonstratebinding in this assay. This is because in the mixture not all bindingsites on the trimers will be bound, thereby leaving extra binding sitesto be captured on the plate. For example, the commercial REMICADEantibody demonstrates trimer binding in this assay.

In contrast, the inventor's antibodies that specifically bind a singleepitope on a sTNF monomer do not demonstrate binding in this assay. Onceeach monomer is bound by the labeled antibody in the mixture, thatepitope is no longer available for binding to the same unlabeledantibody on the plate when the mixture is added to the plate. A sandwichcannot form.

This assay can therefore be used to distinguish between the anti-TNFmonomer-specific antibodies/ligands that bind to A2 or F selectively andthat bind only sTNF monomers and those non-selective commercial andknown anti-TNF antibodies that bind trimers, both tm-TNF and trimericsTNF.

When employed in these assays, the inventor determined that antibodies(both polyclonal or monoclonal) to epitopes A1, A2 or F all were able tobind only sTNF monomers. Results from performance of this assay areshown in FIGS. 3A and 3B.

B. Functional Assay

A functional assay is one that demonstrates the effects of theantibodies on TNF binding cells, such as actinomycin treated WEHI cells.See, e.g., the protocols for “Neutralization of Human TNF Bioactivity”described for anti-human TNF/TNFSF1A antibody by R&D Systems, catalognumber MAB610, clones 28401, pages 1 and 2 (Jun. 17, 2005), incorporatedby reference herein. For the generation of the data in FIGS. 4B and 5,in place of the cells L929, WEHI cells are used. This assay showedinhibition of TNF activity by the selected tested antibodies. In thisassay the antibodies that bind one of the three epitopes on the monomersas described herein are evaluated to determine if they have the abilityto disrupt the formation of bioactive trimers of sTNF and thus inhibitsTNF activity in a dose response curve. Performance of this functionalassay and its results are demonstrated in FIGS. 4B and 5.

C. Results

In the sandwich assay described above, the selective antibodies thatbound one of the three epitopes on the sTNF monomer: A1: SSRTPSDKP, A2:PSDKPVAH or PSDKPVAHV and F: EPIYLGGVF, bound only the monomeric form ofsTNF. As described above, the biotinylated selective anti-TNF antibodiesthat bind one of the epitopes A1, A2 or F would not sandwich with sTNFto cause binding by the same selective non-biotinylated anti-TNFmonomer-specific antibody coated plate. See, e.g., FIGS. 3A and 3B. Incontrast to the results for the anti-A2 and anti-F antibodies/ligands,the commercial anti-TNF REMICADE antibody demonstrated binding in thesandwich assay (see e.g., FIG. 3), showing binding to trimeric TNF.Additionally all polyclonal antibodies to TNF peptides encroaching aninterface region were negative in the sandwich assay, showing lack ofbinding to trimers, demonstrating that binding was restricted tomonomers.

However, only two of the antibodies that exhibited selective binding toone of the three epitopes, A1: SSRTPSDKP, A2: PSDKPVAH and F: EPIYLGGVF,produced dose-responsive inhibition of TNF, suppression of TNFinhibition of cell replication in actinomycin treated WEHI cells in thefunctional assay. See, e.g., FIGS. 4A, 4B and 5. Antibodies to A2 and Fwere capable of disrupting trimer formation and were associated withinhibition of sTNF binding to TNF receptors and inhibition ofcytotoxicity of sTNF on actinomycin treated WEHI cells. These assayfindings provided evidence that the monomer specific antibodies toepitopes A2 and F disrupt trimer assembly by blocking specific aminoacid side chains essential for inter-monomer binding.

Surprisingly, antibodies to the A1 epitope, while monomer specific, wereinactive in blocking TNF function.

Throughout this specification, the words “comprise”, “comprises”, and“comprising” are to be interpreted inclusively rather than exclusively.The words “consist”, “consisting”, and its variants, are to beinterpreted exclusively, rather than inclusively. It should beunderstood that while various embodiments in the specification arepresented using “comprising” language, under various circumstances, arelated embodiment is also be described using “consisting of” or“consisting essentially of” language. It is to be noted that the term“a” or “an”, refers to one or more, for example, “an antibody” isunderstood to represent one or more antibodies. As such, the terms “a”(or “an”), “one or more,” and “at least one” is used interchangeablyherein.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts, which provide one skilled in the art witha general guide to many of the terms used in the present application.All documents listed or referred to herein, including U.S. provisionalapplications Nos. 61/768,044 and 61/756,571, as well as the attached orelectronic Sequence Listing, are incorporated herein by reference.

Given the teachings provided in this specification, one of skill in theart can generate antibodies and other antibody fragments, including highaffinity polyclonal antibodies, affinity purified and humanizedantibodies, monoclonal antibodies and bispecific antibodies that bindspecifically to one or more of the epitopes A2 or F by conventionalmethodologies. Such antibodies and ligands are readily obtained anduseful in the methods disclosed herein. Without further description, itis believed that one of ordinary skill in the art can, using thepreceding description and the following illustrative examples, make andutilize the compositions of the present invention and practice theclaimed methods. While the invention has been described and illustratedherein by references to various specific materials, procedures andexamples, it is understood that the invention is not restricted to theparticular combinations of material and procedures selected for thatpurpose. Numerous modifications and variations of the embodimentsillustrated above are included in this specification and are expected tobe obvious to one of skill in the art. Such modifications andalterations to the compositions and processes described herein arebelieved to be encompassed in the scope of the claims appended hereto.

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What is claimed is:
 1. A monoclonal antibody or a fragment of theantibody that specifically binds to an epitope on a dissociated monomerof human TNF, the epitope having the amino acid sequence EPIYLGGVF,amino acids 116 to 124 of SEQ ID NO: 1, said specific binding disruptingassembly of the monomer into bioactive trimeric human soluble TNF,without binding to intact bioactive trimeric human soluble TNF ortrimeric transmembrane TNF.
 2. The antibody or fragment according toclaim 2, which is a single chain of an antibody, a recombinant antibody,a chimeric antibody, a humanized antibody, a human antibody, or abi-specific antibody.
 3. The antibody or ligand according to claim 2,which is a bi-specific antibody reactive with the TNF monomer-specificepitope having the sequence EPIYLGGVF, amino acids 116 to 124 of SEQ IDNO: 1 and with a TNF monomer-specific epitope having the sequencePSDKPVAH or PSDKPVAHV, amino acids 8-15 or 8-16 of SEQ ID NO:
 1. 4. Theantibody or fragment according to claim 2, wherein said antibodyfragment is an sc-Fv construct, a Fab construct, a Fab₂ construct, or aconstruct containing a light chain or heavy chain variable orcomplementarity determining region (CDR) sequence.
 5. A pharmaceuticalcomposition comprising a monoclonal antibody or fragment thereof thatspecifically binds to an epitope on a dissociated monomer of human TNF,the epitope having the amino acid sequence EPIYLGGVF, amino acids 116 to124 of SEQ ID NO: 1, said specific binding disrupting assembly of themonomer into bioactive trimeric human soluble TNF, without binding tointact bioactive trimeric human soluble TNF or trimeric transmembraneTNF, and a pharmaceutically acceptable carrier or diluent.
 6. Apharmaceutical composition consisting essentially of a monoclonalantibody or antigen-binding fragment thereof that specifically binds toan epitope on a dissociated monomer of human TNF, the epitope having theamino acid sequence EPIYLGGVF, amino acids 116 to 124, said specificbinding disrupting assembly of the monomer into bioactive trimeric humansoluble TNF, without binding to intact bioactive trimeric human solubleTNF or trimeric transmembrane TNF, and a pharmaceutically acceptablecarrier or diluent.
 7. A pharmaceutical composition comprising anantibody or fragment of claim 2, and a pharmaceutically acceptablecarrier or diluent.
 8. A pharmaceutical composition comprising anantibody or fragment of claim 3, and a pharmaceutically acceptablecarrier or diluent.
 9. A pharmaceutical composition comprising anantibody or fragment of claim 4, and a pharmaceutically acceptablecarrier or diluent.
 10. A method for treating a subject having a diseasemediated by human TNF comprising reducing the amount or concentration ofbioactive trimeric sTNF in the blood of a subject having the diseasewithout affecting the amount, concentration or bioactivity of tmTNF bydisrupting or reducing the in vivo assembly or reassembly of dissociatedmonomers of sTNF into bioactive trimeric human sTNF by administering toa subject in need thereof the monoclonal antibody or fragment ofclaim
 1. 11. The method according to claim 10, wherein said disease isrheumatoid arthritis, ankylosing spondylitis, juvenile rheumatoidarthritis, psoriatic arthritis, psoriasis, obesity, metabolic syndrome,atherosclerosis, associated cardiovascular disease, Alzheimer's disease,a neurodegenerative disease, a pathogenic effect of bioactive, trimericsTNF produced during inflammation or during the course of aninflammatory disorder, HIV-1, or type II diabetes.
 12. A method fortreating a subject having a disease mediated by human TNF comprisingreducing the amount or concentration of bioactive trimeric sTNF in theblood of a subject having the disease without affecting the amount,concentration or bioactivity of tmTNF by disrupting or reducing the invivo assembly or reassembly of dissociated monomers of sTNF intobioactive trimeric human sTNF by administering to a subject in needthereof the composition of claim
 5. 13. The method according to claim12, wherein said disease is rheumatoid arthritis, ankylosingspondylitis, juvenile rheumatoid arthritis, psoriatic arthritis,psoriasis, obesity, metabolic syndrome, atherosclerosis, associatedcardiovascular disease, Alzheimer's disease, a neurodegenerativedisease, a pathogenic effect of bioactive, trimeric sTNF produced duringinflammation or during the course of an inflammatory disorder, HIV-1, ortype II diabetes.